The Role Of NLRP3 Inflammasome In Contrast Induced Acute Kidney Injury

AimsContrast induced acute kidney?CI-AKI?is the third cause of hospital-acquired acute kidney injury.Various mechanisms involved in the CI-AKI process,the present study reveal several of them 1,renal hemodynamics and ischemia caused by hypoxia;2,local kidney oxidative stress;3 contrast agent toxic effects on the renal tubular epithelial cells.4,role of some endothelin.Nucleotide-binding oligomerization domain receptor-like protein 3?NLRP3?is a newly discovered pattern recognition receptors expressed in a variety of intrinsic renal cells such as renal tubular epithelial cells,podocytes,mesangial cells.After activated,it formed NLRP3inflammasome with apoptosis-associated speck-like protein?apoptosis-associated speck-like protein containing CARD,ASC?,then activating caspases 1?caspase-1?,causing interleukin 18?IL-18?and interleukin-1??IL-1??mature and release.This research focus on several questions below1.the effect of contrast on human kidney epthelial cells.2.the function of NLRP3 inflammasome in the process of contrast induced HK-2 cell injury.3.design a CI-AKI model.4.NLRP3 gene function in CI-AKI mouse.MethodsHuman kidney 2?HK-2?cells were cultured in DMEM-F12 medium with 5%FBS.The cells were seeded at 1.5?106 cells/10 cm diameter dish for 2-3 days.For contrast treatments,cells were incubated in FBS free DMEM-F12 medium for 24 h.The following day,contrast medium?Omnipaque?350 mgI/ml were added to the culture media?final concentration 20-40 mgI/ml?,then HK-2 cells were treated with the mixture of FBS free culture media and contrast for 72 h.Cells were divided into control group,Omnipaque groups?treated with the mixture of FBS free culture media and contrast?,NLRP3-siRNA+Omnipaque group?transfected with NLRP3 siRNA,then treated with the mixture of FBS free culture media and contrast 24h later?,ASC-siRNA+Omnipaque group?transfected with ASC siRNA,then treated with the mixture of FBS free culture media and contrast 24h later?and mannitol group?treated with the mixture of FBS free culture media and mannitol as the mixture's osmolarity equal to O group?.The effects of contrast and NLRP3inflammasome deficiency on cell apoptosis were detected by flow cytometry.NLRP3 inflammasome related proteins NLRP3,ASC,caspase 1 and apoptosis regulatory proteins including caspase 3,caspase8,BCL-2 and Bax were detected by Western blotting.NLRP3 and ASC mRNA levels were detected by real-time PCR.We use C57BL/6 mice and NLRP3^-/-to design CI-AKI mouse model,In the research,we use unilateral nephrectomy,furosemide,dehydration as pretreatments.NLRP3 inflammasome related proteins NLRP3,ASC,caspase 1 and apoptosis regulatory proteins including caspase 3,caspase8,BCL-2 and Bax were detected by Western blotting.Kidney injury is measured by HE stain.The effects of contrast and NLRP3 inflammasome deficiency on cell apoptosis were detected by TUNEL.NLRP3inflammasome related proteins NLRP3,ASC were detected by Immunohistochmeistry.ResultsThe first part of this study explores the effect of contrast agent on human renal tubular epithelial?human kidney-2 HK-2?cells on NLRP3expression and cell apoptosis.The results showed that iohexol stimulation cause NLRP3,ASC mRNA increased?p<0.05?in HK-2 cells,iodixanol and mannitol had no apparent effect.Further studies showed that iohexol could induce HK-2 cells apoptosis,the effect is dose-dependent manner?p<0.05?.And iohexol can induce HK-2 cell viability decreased?p<0.05?.The results showed that:hypotonic contrast agent iohexol induce HK-2 cells can apoptosis,intracellular NLRP3,ASC mRNA increased.The osmotic pressure is not caused by the above-mentioned increase in mRNA reasons.The second part of the research studies the role of inflammasome in the process of contrast induced human renal tubular epithelial cell injury.The results showed that iohexol stimulation can significantly induce,apoptotic protein BAX,caspase-3/cleaved caspase-3,caspase-8/cleaved caspase-8 content increased?p<0.05?in HK-2 cells.Anti-apoptotic protein BCL-2 decreased?p<0.05?.Meanwhile iohexol induced NLRP3 inflammasome-associated protein NLRP3,ASC,cleaved caspase-1 content increase.Using SiRNA to silence NLRP3 and ASC mRNA can block iohexol induced HK-2 cells NLRP3 inflammasome pathway activation and downstream IL-18 and IL-1?release?p<0.05?.At the same time,contrast-induced apoptotic protein BAX,caspase-3/cleaved caspase-3,caspase-8/cleaved caspase-8 content increase and the anti-apoptotic protein BCL-2 decreased significantly reduced?p<0.05?.Reduce.The results showed that:hypotonic contrast agent iohexol can activate HK-2 cells NLRP3 inflammasome pathway,silencing NLRP3 or ASC gene can block inflammasome activation.Hypotonic contrast agent iohexol induced apoptosis in HK-2 cells,silencing NLRP3 or ASC gene reduce apoptosis.The third part discusses the preparation of CI-AKI mouse model.Kidney disease experts have create variety of CI-AKI models.A simple injection of contrast agent cannot manufacture CI-AKI model efficiently,Using a variety of pretreatment methods before injection of contrast agent can significantly improve modeling success rate.Currently used Pretreatment methods are:unilateral nephrectomy,injection diuretics,renal vascular occlusion row ischemia-reperfusion injury,dehydration,injection of prostaglandin.In this experiment,we found that:unilateral nephrectomy+furosemide+24h dehydration relatively a good modeling method.By real-time PCR technology,we found iohexol induce NLRP3and ASC mRNA increased?p<0.05?.The results showed that:unilateral nephrectomy+24h+furosemide dehydration 10ul/g+iohexol 10ul/g is a good hypotonic CI-AKI mouse model methods.The fourth part of this study explores the role NLRP3 in CI-AKI mouse model.The results showed that iohexol significantly stimulate apoptotic protein BAX,caspase-3/cleaved caspase-3,caspase-8/cleaved caspase-8 content increased and Anti-apoptotic protein BCL-2decreased?p<0.05?.Tunel staining showed iohexol induce renal tubular cell apoptosis?p<0.05?.Meanwhile iohexol induce inflammasome assocated protein NLRP3,ASC,cleaved caspase-1 content increased;contrast-induced apoptotic protein BAX,caspase-3/cleaved caspase-3,caspase-8/cleaved caspase-8 content increase and the anti-apoptotic protein BCL-2 decreased significantly reduced?p<0.05?in NLRP3^-/-mice;Tunel staining showed renal tubular cell apoptosis decrease?p<0.05?in NLRP3^-/-mice;HE staining showed iohexol induce mouse kidney tissue injury,NLRP3^-/-mouse renal tissue is mitigated in NLRP3 mice.Immunohistochemical showed that compared with the control group,iohexol induce NLRP3 and ASC expression in WT mice.T The results showed that::NLRP3 inflammasome pathway is activated in WT mice;renal tissue injury,tubular cell apoptosis and renal structural damage are observed;NLRP3^-/-mice suffers less kidney injury than WT mice.ConclusionIn summary,the following conclusions can be drawn from this study:1,contrast can cause apoptosis of HK-2 cells;2,low permeability contrast agent iohexol can activate NLRP3 inflammasome pathway in HK-2 cells,silencing NLRP3 can reduce contrast-induced apoptosis;3,unilateral nephrectomy+24h+furosemide dehydration 10ul/g+iohexol is a preferred method for making low permeability contrast CI-AKI mouse model.4,NLRP3^-/-mice are better than WT mice to resist the kidney damage caused by CI-AKI;5,NLRP3 inflammasome participates and promotes the pathogenesis of CI-AKI.